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EC number: 202-705-0
CAS number: 98-83-9
Intravenous Study for Urinary Metabolite
Intravenous doses of alpha-methylstyrene (10 mg/kg)
were mainly excreted in the urine with 76 +/- 2 % excreted in the first
24 hours postdosing and 86 +/- 1 % by 72 hours. Faecal elimination
accounted for 2 % of the dose. Exhalation of volatile organics and
carbon dioxide accounted for only 2 % and 0.02 % of the dose,
respectively. Concentrations of alpha-methylstyrene equivalents were
low, and only 0.3 % of the radioactivity was recovered in the tissues.
The concentration of alpha-methylstyrene and/or its metabolites in blood
was 16 ng Eq per gram.
The profiles of metabolites present following an
intravenous dose of alpha-methylstyrene (10 mg/kg) were determined for
each urinary collection interval up to 48 hours postdosing for one rat.
The peak eluting at 10:25 (metabolite D) coelutes with atrolactic acid,
and over 20 % of the administered dose was excreted as this metabolite.
Metabolite E was most abundant in the early urine collection (0 to 6
hours), with considerably less detected in later collections, suggesting
that it may be an early intermediate metabolite of alpha-methylstyrene.
Oral Study for Urinary Metabolite Identification
Alpha-methylstyrene was administered orally to one
rat at a dose of 1000 mg/kg in an effort to obtain as much urinary
metabolite as possible. Urine from this experiment was chromatographed
and gave a similar profile as the urine from the intravenous study. Five
of the metabolite peaks were identified by GC/MS. Metabolite B was
treated with beta-glucuronidase, and the aglycone was analyzed by GC/MS.
The spectrum of the bis-trimethylsilane derivative of a 2-phenyl-1,2
-propanediol standard had no M+ at 296 but had peaks at m/z 281 (loss of
a methyl), 193 (loss of CH2O-trimethylsilane ), and 147. The spectra of
metabolite C and the aglycone of metabolite B were virtually identical
to that of the bis-trimethylsilane derivative of a
2-phenyl-1,2-propanediol standard. Therefore, it was concluded that
metabolite B is a glucuronide of 2-phenyl-1,2-propanediol and that
metabolite C is 2-phenyl-1,2-propanediol.
Metabolite B was analyzed by 13C-DEPT NMR in an
effort to determine the position of attachment of the glucuronide group
in the conjugate. Of the two oxygenated carbon atoms, only the terminal
methylene group had protons with which the pulsed carbon nucleus could
relax. Therefore, the position of the methylene resonance was determined
in these characterization experiments using NMR. The spectra displayed a
doublet for each carbon bearing a proton, possibly indicating that this
metabolite was present as a pair of diasteriomers. The 13C-DEPT NMR
spectrum of 2-phenyl-1,2-propanediol was determined in order to
interpret the spectra for metabolite B, and the methylene resonance
appeared at 72 ppm. If the glucuronide conjugate were attached to the
carbon alpha to the phenyl ring, the chemical shift of the methylene
carbon would have been approximately 3 ppm upfield (at 69 ppm) relative
to that for the unconjugated 2-phenyl-1,2-propanediol; had it been at
the beta carbon, the shift would have been approximately 10 ppm
downfield (at 82 ppm). In the 13C-DEPT NMR spectrum of metabolite B, the
methylene resonances were at 79 ppm, consistent with the attachment of
the glucuronide moiety to the carbon beta to the phenyl ring.
A bis-trimethylsilane derivative of an atrolactic
acid standard and metabolite D were analyzed by GC/MS, and the spectra
were identical. A standard of 2 -phenylpropionic acid and metabolite F
were analyzed by GC/MS, and both spectra contained a molecular ion at
m/z 150 and signals at m/z 105 (loss of COOH) and 77. Metabolite E was
treated with acylase, and the incubation mixture was chromatographed as
described previously for isolation of the peak. The profile was compared
with the HPLC profile of the untreated urine. Treatment with acylase
converted metabolite E to a new component that eluted between
metabolites A and B. The 1H NMR spectrum of metabolite E was consistent
with a N-acetylcysteine conjugate resulting from reaction of glutathione
with the epoxide of alpha-methylstyrene, followed by further metabolism
to the mercapturate. The 13C-DEPT NMR also corroborated this finding.
The 13C chemical shift of the methylene group beta to the ring was
consistent with attachment of the sulphur atom to this carbon atom.
Trimethylsilane derivatives of the mercapturate(s) were prepared for
analysis by GC/MS. Two di-derivatized and two tri-derivatized products
were present in roughly equal amounts. The fragmentation patterns (in
particular the ion at 193 a.m.u.) in these spectra were also consistent
with the formation of just one of two possible positional isomers for
the mercapturate. The NMR and mass spectral data indicated that a
diasteriomeric pair of mercapturates was formed as metabolites.
The presence of roughly equal amounts of the
diasteriomeric mercapturates suggests that the initial epoxidation of
alpha-methylstyrene is not sterioselective and proceeds with no marked
preference for the antarafacial or suprafacial addition of active oxygen
to yield enantiomeric epoxides. Both enzymatic hydrolysis and
glutathione conjugation of epoxides are known to proceed by SN2
reactions. Therefore, enzymatic hydrolysis can yield enantiomeric diols.
Further oxidation of the terminal hydroxy group of these diols to form
atrolactic acid does not affect the chiral centre at the benzyl
position, and the potential products are enantiomers. However,
conjugation with a chiral molecule such as glutathione or glucuronic
acid would produce diasteriomeric metabolites from the enantiomeric
products, as is the case with the mercapturates and glucuronides
characterized in these studies.
The time weighted average alpha-methylstyrene
vapour concentrations during exposure for the 300 and 900 ppm groups
were 304 and 900 ppm. Animals exposed to 300 or 900 ppm received mean
doses of 130.8 and 340.1 mg/kg, or 26.63 and 20.05 uCi, respectively.
For the 300 ppm group, urinary excretion was the
main route of elimination, comprising 88.2 % of the absorbed dose, with
volatile breath and faeces accounting for 3.1 and 2.2 %, respectively.
These trends were consistent for all collection intervals. Over 90 % of
the absorbed dose was eliminated within 48 hours post-initiation of
exposure. The same pattern of elimination was observed in the 900 ppm
group; 92.4 % was excreted in urine, with volatile breath and faeces
accounting for 2.5 and 2.6 %, respectively.
Tissue distribution of radioactivity at 6, 24 (300
ppm only), and 72 hrs after initiation of inhalation: 2.6 – 10.1 % and
1.1 – 2.4 % of the inhaled radiolabeled alpha-methylstyrene was
recovered in the residual carcass and tissues in the 300 and 900 ppm
groups, respectively. The highest concentrations of radiolabeled
alpha-methylstyrene (ug Eq/g tissue) were observed in adipose, bladder,
liver, kidney, and skin at 6, 24 (300 ppm only), and 72 hours after
initiation of exposure. This is consistent with the lipophilic nature of
alpha-methylstyrene and the fact that most of the dose was eliminated in
urine. Elevated levels of radiolabel were present in the small intestine
compared to the stomach and large intestine, suggesting biliary
excretion and re-absorption of [14C]alpha-methylstyrene-derived
metabolite(s). Tissues for the 24-hour time point in the 300 ppm group
were removed from animals used for serial blood sampling.
The metabolite profile observed in urine from 300
and 900 ppm male rats was qualitatively the same as that in the
intravenous study. Much more atrolactic acid was observed in urine
collected during the first 24 hrs in the inhalation studies compared to
the intravenous study. Rats exposed to 900 ppm exhibited nearly a
twofold increase in excretion of atrolactic acid, accompanied by a
corresponding drop in excretion of 2-phenyl-1,2-propanediol glucuronide,
in urine collected between 12 and 24 hrs compared to rats exposed to 300
Characterization of the 14C Profile in Blood
In extraction method development experiments,
recovery of carbon-14 from blood spiked with [14C]alpha-methylstyrene
was 95 +/- 6 %. Recoveries of carbon-14 from blood samples obtained from
the 300 and 900 ppm groups were 74 +/- 10 % and 82 +/- 9 %,
respectively. These recoveries suggest sequestration of metabolites by
red blood cells. Chromatographic analysis of red blood cell lysate
showed that no radioactivity was associated with heme.
Alpha-methylstyrene concentrations in blood dropped
precipitously in the 300 ppm rats upon cessation of exposure, from more
than 6 ug/mL just prior to termination of exposure (5.5 hrs) to an
average of 0.97 ug/mL at 7 hrs. From 7 – 24 hrs, alpha-methylstyrene
concentrations in blood decreased at a much slower rate.
Alpha-methylstyrene concentration in blood dropped from more than 24
ug/mL at 5.5 hrs into the exposure to ca. 10 ug/mL in the first hour
after cessation of exposure in the 900 ppm rats.
Four metabolites were extracted from blood obtained
in the inhalation study. The major component at all time points was
2-phenyl-1,2-propanediol. Atrolactic acid, 2 -phenylpropionic acid, and
an additional radiolabeled component noted as blood metabolite 1 were
also observed. Identities of atrolactic acid, 2-phenyl-1,2 -propanediol,
2-phenylpropionic acid, and alpha-methylstyrene peaks were established
by co-elution with non-radiolabeled standards.
Male F344/N rats were exposed to alpha-methylstyrene via
intravenous or nose-only inhalation exposure. In both studies, the
substance was eliminated primarily in the urine (approximately 90 %)
within 72 hours, with volatile breath and feces accounting for only a
small amount (1 - 3 %) of elimination. In the inhalation study, the
elimination half-life was calculated at 3 to 5 hours, with the highest
concentrations of alpha-methylstyrene-derived radioactivity retained in
the adipose tissue, urinary bladder, liver, kidney, and skin. Following
intravenous dosing, the kidney, heart, lung, liver, urinary bladder, and
spleen retained the highest concentrations of radioactivity. In both the
intravenous study and the inhalation study, the major urinary
metabolites of alpha-methylstyrene were the glucuronide conjugate of 2
-phenyl-1,2-propanediol and atrolactic acid. In the inhalation study,
the major metabolites in the blood were 2-phenyl-1,2-propanediol and
Based on these studies, the proposed metabolic pathway
for alpha-methylstyrene involves an initial non-stereoselective
epoxidation followed by hydrolysis to form 2-phenyl-1,2-propanediol
followed by either oxidation to atrolactic acid or formation of the
glucuronide conjugate, conjugation with glutathione and subsequent
cleavage to the mercapturate, or rearrangement to form an aldehyde that
is oxidized to yield 2-phenylpropionic acid. The dose-dependent
pharmacokinetic parameters coupled with decreased excretion of
2-phenyl-1,2-propanediol glucuronide at 900 ppm indicate that
glucuronide formation was saturated at this dose.
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